Sarcoglycan (SG) complex is a membrane-spanning glycoprotein complex composed of four dystrophin-associated proteins (DAPs) and its absence or markedly reduced expression due to mutation of any one of the SG gene causes a group of Duchenne-like muscular dystrophies. The SG complex is thus considered to be essential for skeletal muscle survival, but we have not well understood its functions. The possibility has been suggested that the SG complex participates in signaling processes as a receptor. On the other hand, α-syntrophin (Syn) has been shown to be associated with the signaling protein nNOS. Recently, the gene knockout mice of α-dystrobrevin (DB) were found to exhibit a muscular dystrophy phenotype, and shoed secondary loss of nNOS from the sarcolemma.To understand the functions of the SG complex such as its putative signaling function, it is essential to clarify the structural relationship of the SG complex with the other members of dystrophin-DAP complex and determine, if present, the link between the SG complex and the intracellular signaling molecules like nNOS. In this study, we developed new methods for preparing various novel DAP complexes containing the SG complex by heat treatment of the purified dystrophin-DAP complex. Bu the analysis of these novel complexes, we could demonstrate that the SG complex is associated with sarcospan (SPN) and that this SG-SPN complex interacts, on the one hand, with the dystroglycan complex, and on the other, with Syn and/or DB. Further analysis of the last complex revealed that the N-terminal half of α-DB participates in this association. It is thus considered that the SG-SPN complex is linked to the signaling protein nNOS via α-Syn fixed to α-DB and regulates the activity of nNOS.